1. Field of the Invention
The present invention generally relates to engineered vaults that are free of cellular debris, methods of making the engineered vaults, and methods of packaging passenger molecules in the engineered vaults.
2. Description of the Related Art
Vaults are cytoplasmic ubiquitous ribonucleoprotein particles first described in 1986 that are found in most eukaryotic cells. See Kedersha & Rome (1986) J Cell Biol 103(3):699-709. Native vaults are about 12.9±1 MDa ovoid spheres with overall dimensions of about 72 nm×42 nm×42 nm. Vaults have been recombinantly produced using a baculovirus expression system and heterologous proteins have been encapsulated therein as fusion proteins, i.e., heterologous proteins recombinantly fused to the major vault protein interaction domain (mINT) of vault polyADP-ribose polymerase (VPARP). See Stephen, et al. (2001) J Biol Chem 276(26):23217-23220, and Kickhoefer, et al. (2005) PNAS USA 102(12):4348-4352. Specifically, empty vaults are recombinantly produced using Sf9 insect cells and a baculovirus vector and then the empty vaults are mixed with passenger-mINT fusion proteins (passenger molecules recombinantly fused to mINT), whereby the passenger-mINT fusion proteins become localized inside the vaults.
Unfortunately, the prior art methods of making vaults and packaging passenger molecules inside of the vaults have several drawbacks. First, the use of Sf9 insect cells and viral vectors to make the empty vaults results in contaminating proteins, e.g., viral vector contaminants such as baculovirus proteins, which are difficult, if not impossible to separate from vaults. These contaminant proteins may result in adverse or undesired immunogenic reactions when administered to a subject and/or interfere with the activity or function of the passenger molecules. Second, not all molecules, e.g., nucleic acid molecules, small molecules, etc., can be recombinantly fused to mINT. Thus, not all molecules can be packaged inside of vaults using the INT-fusion method. In fact, some proteins, e.g., the small (67 amino acid) peptide HIV-1 Gag 148-214, do not package efficiently, if at all, using INT-fusion method.
Therefore, a need exists for methods of making empty vaults that are free of contaminant proteins and methods of packaging a variety of passenger molecules, including molecules that cannot be recombinantly fused to mINT inside the vaults.